bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion 2 morphogenesis 3 4 Short title: Key role for HIV-1 integrase-RNA interactions in virion maturation 5 6 Jennifer Elliott1, Jenna E. Eschbach1, Pratibha C. Koneru2, Wen Li3,4, Maritza Puray 7 Chavez1, Dana Townsend1, Dana Lawson1, Alan N. Engelman3,4, Mamuka Kvaratskhelia2, 8 Sebla B. Kutluay1 9 10 1 Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, 11 MO 63110, USA 12 2 Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, CO 80045 13 3 Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 14 02215 15 4 Department of Medicine, Harvard Medical School, Boston, MA 02115 16 17 18 Correspondence: [email protected] 19 20 21 22 23 24 25 26 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 27 ABSTRACT 28 A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit 29 pleotropic effects during virus replication. However, the underlying mechanism for the class II 30 phenotype is not known. Here we demonstrate that all tested class II IN substitutions 31 compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly 32 reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely 33 affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) 34 directly compromising IN-RNA interactions without substantially affecting IN levels or functional 35 IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral 36 ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of 37 the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for 38 the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion 39 maturation. 40 41 INTRODUCTION 42 Infectious HIV-1 virions are formed in a multistep process coordinated by interactions 43 between the HIV-1 Gag and Gag-Pol polyproteins, and the viral RNA (vRNA) genome. At the 44 plasma membrane of an infected cell, Gag and Gag-Pol molecules assemble around a vRNA 45 dimer and bud from the cell as a spherical immature virion, in which the Gag proteins are 46 radially arranged [1-3]. As the immature virion buds, the viral protease enzyme is activated and 47 cleaves Gag and Gag-Pol into their constituent domains triggering virion maturation [1, 2]. 48 During maturation the cleaved nucleocapsid (NC) domain of Gag condenses with the RNA 49 genome and pol-encoded viral enzymes [reverse transcriptase (RT) and integrase (IN)] inside 50 the conical capsid lattice, composed of the cleaved capsid (CA) protein, which together form the 51 core [1-3]. 2 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 52 After infection of a target cell, RT in the confines of the reverse transcription 53 complex (RTC) synthesizes linear double stranded DNA from vRNA [4]. The vDNA is 54 subsequently imported into the nucleus, where the IN enzyme catalyzes its insertion into the 55 host cell chromosome [5, 6]. Integration is mediated by the intasome nucleoprotein complex that 56 consists of a multimer of IN engaging both ends of linear vDNA [7]. While the number of IN 57 protomers required for intasome function varies across Retroviridae, single particle cryogenic 58 electron microscopy (cryo-EM) structures of HIV-1 and Maedi-visna virus indicate that lentivirus 59 integration proceeds via respective higher-order dodecamer and hexadecamer IN arrangements 60 [8, 9], though a lower-order intasome comprised of an HIV-1 IN tetramer was also resolvable by 61 cryo-EM [9]. 62 A number of IN substitutions which specifically arrest HIV-1 replication at the integration 63 step have been described [10]. These substitutions are grouped into class I to delineate them 64 from a variety of other IN substitutions, which exhibit pleiotropic effects and are collectively 65 referred to as class II substitutions [10-12]. Class II IN substitutions or deletion of entire IN 66 impair proper particle assembly [11, 13-25], morphogenesis [11, 15, 21-23, 26-28] and reverse 67 transcription in target cells [10, 11, 17, 19-21, 23, 25-44], in some cases without impacting IN 68 catalytic function [15, 16, 19, 20, 30, 31, 34, 36, 45-47]. A hallmark morphological defect of 69 these viruses is the formation of aberrant viral particles with viral ribonucleoprotein (vRNP) 70 complexes mislocalized outside of the conical CA lattice [11, 15, 21-23, 26-28]. Strikingly similar 71 morphological defects are observed in virions produced from cells treated with allosteric 72 integrase inhibitors (ALLINIs, also known as LEDGINs, NCINIs, INLAIs or MINIs) [26, 27, 48- 73 55]. ALLINIs induce aberrant IN multimerization in virions by engaging the V-shaped pocket at 74 the IN dimer interface, which also provides a principal binding site for the host integration 75 targeting cofactor lens epithelium-derived growth factor (LEDGF)/p75 [50, 54, 56-60]. The 76 recent discovery that HIV-1 IN binds to the vRNA genome in virions and that inhibiting IN-RNA 3 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 77 interactions leads to the formation of eccentric particles provided initial clues about the role of IN 78 during virion morphogenesis [28]. 79 HIV-1 IN consists of three independently folded protein domains: the N-terminal domain 80 (NTD), catalytic core domain (CCD), and C-terminal domain (CTD) [7, 61], and vRNA binding is 81 mediated by a constellation of basic residues within the CTD [28]. However, class II IN 82 substitutions are located throughout the entire length of the IN protein [10, 12], which raises the 83 question as to how these substitutions impair virus maturation. The structural basis for IN 84 binding to RNA is not yet known; however, in vitro evidence indicates that IN binds RNA as 85 lower-order multimers, and conversely RNA binding may prevent the formation of higher order 86 IN multimers [28]. Notably, aberrant IN multimerization underlies the inhibition of IN-RNA 87 interactions by ALLINIs [28] and subsequent defects in virion maturation [26-28, 48, 49, 51-55]. 88 Therefore, it seems plausible that class II IN substitutions may exert their effect on virus 89 replication by adversely affecting functional IN multimerization. However, a systematical 90 evaluation of the effects of IN substitutions on IN multimerization, IN-RNA binding, and virion 91 morphology is lacking. As such, it remains an open question how functional IN multimerization 92 and/or IN-RNA interactions influence correct virion morphogenesis. 93 Eccentric virions generated via class II IN substitutions or ALLINI treatment are defective 94 for reverse transcription in target cells [10, 11, 17, 19-21, 23, 25-44, 48, 49, 51, 54, 58, 62] 95 despite containing equivalent levels of RT and vRNA genome as wild type (WT) particles [26, 96 63]. In addition, neither the condensation of the viral genome by NC [26, 63] nor its priming [63] 97 appear to be affected. We and others have recently shown that premature loss of the viral 98 genome and IN, as well as spatial separation of RT from vRNPs, may underlie the reverse 99 transcription defect observed in eccentric viruses generated in the presence of ALLINIs or the 100 class II IN R269A/K273A substitutions [59, 64]. These findings support a model in which the 101 capsid lattice or IN binding to vRNA itself is necessary to protect viral components from the host 4 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 102 environment upon entering a target cell. Whether the premature loss of the viral genome and IN 103 is a universal outcome of other class II IN substitutions is unknown. 104 In this work, we aimed to determine the molecular basis of how class II IN substitutions 105 exert their effects on HIV-1 replication. In particular, by detailed characterization of how class II 106 substitutions impact IN multimerization, IN-RNA interactions and virion morphology, we aimed to 107 dissect whether loss of IN binding to vRNA or aberrant IN multimerization underlies the 108 pleiotropic defects observed in viruses bearing class II IN mutations.